The receptors that mediate chemotaxis by Escherichia coli are the best-characterized members of a large family of bacterial chemoreceptors and of a larger family of prokaryotic and eukaryotic sensory receptors. This proposal focuses on sensory transduction by these receptors, with emphasis on structure, signaling and interactions with other sensory components and in higher order assemblies. The proposed work combines biochemical, biophysical, genetic and structural approaches. Several aspects involve continuing collaborations with prominent biophysicists and an interactive cooperation with a molecular modeler. Over the past 8 years, we have made extensive use of cysteine substitutions and sulfhydryl chemistry. This strategy will again be utilized extensively. In the current funding period, we obtained indications that the transmembrane domain of chemoreceptors was notably dynamic and loosely packed. We will investigate this unexpected observation by an array of biochemical, biophysical and modeling approaches. The hydrophobic boundaries of chemoreceptor transmembrane helices are only vaguely defined, as is the case for most membrane proteins. Because the issue has implications for both structure and signaling, we will purse it, using multiple strategies. Our use of diagnostic cross-linking between cysteine pairs in the membrane has proved notably informative in detecting the conformational change of chemoreceptor transmembrane signaling. We propose to extend and exploit this strategy to learn more about conformational signaling induced by ligand binding and to identify the compensatory conformational changes driven by adaptational modifications. Recently, we discovered that the methylesterase bound the same interaction site on the carboxyl-terminus of chemoreceptors as the methyltransferase. However, the mechanisms by which these interactions enhance activity of the adaptational enzymes appear quite different. We propose to investigate this in detail. Different chemoreceptors in E. coli are not equivalent. In the current funding period we defined the best-documented example of receptor interaction and cooperation by characterizing the mode of action of "low-abundance" receptor Trg. We propose to continue characterization of structural and functional features of this receptor, and to use the phenomenon of adaptational assistance of low-abundance receptors by high-abundance receptors as a tool for investigating the tantalizing notion that receptor clustering, cooperation and interaction are fundamental to the mechanisms of chemotaxis. We will determine cellular dosages of chemotaxis components to define the limits of signaling complex stoichiometries. Our multi-faceted, multi-disciplinary approach promises to reveal new information about the structures, interactions and conformational changes that transmembrane proteins use to transduce, transmit and integrate sensory information.